In recent years, the development of genetic engineering and the resulting sufficiency of information on three-dimensional protein structures, genome sequences, and the like have made it possible to create a protein with a new function by artificially altering a protein or, specifically, to create a protein with new activity by altering, based on a protein having certain activity, some amino-acid residues of the protein. For alteration of amino-acid residues to other amino-acid residues, natural amino acids are limited as options, and as such, may make it difficult to produce a protein having a desired function and desired activity.
Proposed in view of this as a method for expanding the functions of a protein are various methods that involves the introduction of an unnatural amino acid into a protein (e.g., see Wang, L., and Schultz, P. G., Expanding the genetic code. Angew Chem Int Ed Engl, 2005, 44, 34-66.). Among them, a method that involves the use of an aminoacyl-tRNA synthetase (hereinafter referred to sometimes as “aaRS”) mutant has recently been developed as a method that can be used in living cells with high yields (International Publication No. 2003/014354 Pamphlet (published on Feb. 20, 2003), Lee, N., Bessho, Y., Wei, K., Szostak, J. W., and Suga, H., Ribozyme-catalyzed tRNA aminoacylation. Nat Struct Biol, 2000, 7, 28-33.).
An aaRS exists in all living organisms. An aaRS is responsible for faithfully translating a genetic code by accurately associating an amino acid with tRNA. A reaction catalyzed by an aaRS includes a first step of activating an amino acid with ATP and a second step of adding an activated aminoacyl adenylate intermediate to the 3′-end of tRNA. Both of the reactions are carried out at a single active site (aminoacylation active site) (Fersht, A. R., and Kaethner, M. M., Mechanism of aminoacylation of tRNA. Proof of the aminoacyl adenylate pathway for the isoleucyl- and tyrosyl-tRNA synthetases from Escherichia coli K12. Biochemistry, 1976, 15, 818-823; and Freist, W., and Sternbach, H., Tyrosyl-tRNA synthetase from baker's yeast. Order of substrate addition, discrimination of 20 amino acids in aminoacylation of tRNATyr-C-C-A and tRNATyr-C-C-A(3′NH2). Eur J Biochem, 1988, 177, 425-433.).
There are two classes of aaRS, each with its own origin of evolution, and each of the classes includes 10 aaRSs (Eriani, G., Delarue, M., Poch, O., Gangloff, J., and Moras, D., Partition of tRNA synthetases into two classes based on mutually exclusive sets of sequence motifs. Nature, 1990, 347, 203-206.). A class I aaRS has an aminoacylation active site called a Rossman-fold domain. Meanwhile, a class II aaRSs does not have a Rossman-fold domain, but has an aminoacylation active site surrounded by an antiparallel beta-sheet. An aaRS must associate an amino acid with tRNA accurately. tRNAs are large molecules whose molecular weight exceeds 20,000, and vary in sequence. Meanwhile, amino acids are small molecules that share an α-amino group and an α-carboxyl group as common structures, and differ only in side chain. This makes it more difficult for an aaRS to discriminate between amino acids than to discriminate between tRNAs. For example, isoleucine and valine differ solely in methyl group, and it is believed to be difficult for an enzyme to recognize the difference (Pauling, L., The Probability of Errors in the Process of Synthesis of Protein Molecules, 1957.).
For this reason, each of seven types of natural aaRS, which amount to one third of all the natural aaRSs, namely an isoleucyl-tRNA synthetase (hereinafter referred to as “IleRS”) of class I, a valyl-tRNA synthetase (hereinafter referred to as “ValRS”) of class I, a leucyl-tRNA synthetase (hereinafter referred to as “LeuRS”) of class I, an alanyl-tRNA synthetase (hereinafter referred to as “AlaRS”) of class II, a prolyl-tRNA synthetase (hereinafter referred to as “ProRS”) of class II, a threonyl-tRNA synthetase (hereinafter referred to as “ThrRS”) of class II, and a phenylalanyl-tRNA synthetase (hereinafter referred to as “PheRS”) of class II, is known to have an aminoacylation active site, serving as an active site, which not only recognizes a correct substrate amino acid but also misrecognizes another amino acid similar the correct substrate amino acid (Crepin, T., Yaremchuk, A., Tukalo, M., and Cusack, S., Structures of two bacterial prolyl-tRNA synthetases with and without a cis-editing domain. Structure, 2006, 14, 1511-1525.; Dock-Bregeon, A., Sankaranarayanan, R., Romby, P., Caillet, J., Springer, M., Rees, B., Francklyn, C. S., Ehresmann, C., and Moras, D., Transfer RNA-mediated editing in threonyl-tRNA synthetase. The class II solution to the double discrimination problem. Cell, 2000, 103, 877-884.; Fukai, S., Nureki, O., Sekine, S., Shimada, A., Tao, J., Vassylyev, D. G., and Yokoyama, S., Structural basis for double-sieve discrimination of L-valine from L-isoleucine and L-threonine by the complex of tRNA (Val) and valyl-tRNA synthetase. Cell, 2000, 103, 793-803.; Fukunaga, R., and Yokoyama, S., Aminoacylation complex structures of leucyl-tRNA synthetase and tRNALeu reveal two modes of discriminator-base recognition. Nat Struct Mol Biol, 2005, 12, 915-922.; Lin, L., Hale, S. P., and Schimmel, P., Aminoacylation error correction. Nature, 1996, 384, 33-34.; Nomanbhoy, T. K., Hendrickson, T. L., and Schimmel, P., Transfer RNA-dependent translocation of misactivated amino acids to prevent errors in protein synthesis. Mol Cell, 1999 4, 519-528.; Nureki, O., Vassylyev, D. G., Tateno, M., Shimada, A., Nakama, T., Fukai, S., Konno, M., Hendrickson, T. L., Schimmel, P., and Yokoyama, S., Enzyme structure with two catalytic sites for double-sieve selection of substrate. Science, 1998, 280, 578-582.; Roy, H., Ling, J., Irnov, M., and Ibba, M., Post-transfer editing in vitro and in vivo by the beta subunit of phenylalanyl-tRNA synthetase. EMBO J, 2004, 23, 4639-4648.; Ruan, B., and Soll, D., The bacterial YbaK protein is a Cys-tRNAPro and Cys-tRNA Cys deacylase. J Biol Chem, 2005, 280, 25887-25891.; Sokabe, M., Okada, A., Yao, M., Nakashima, T., and Tanaka, I., Molecular basis of alanine discrimination in editing site. Proc Natl Acad Sci USA, 2005, 102, 11669-11674.; and Swairjo, M. A., Otero, F. J., Yang, X. L., Lovato, M. A., Skene, R. J., McRee, D. E., Ribas de Pouplana, L., and Schimmel, P., Alanyl-tRNA synthetase crystal structure and design for acceptor-stem recognition. Mol Cell, 2004, 13, 829-841.). As a result of such misrecognition, an aminoacyl adenylate intermediate is produced by activation with an amino acid different from the correct substrate, or aminoacyl tRNA is produced as a product thereof.
However, each of these aaRSs has an editing reaction active site separate from the aminoacylation active site, and as such, has activity to hydrolyze the mistakenly produced aminoacyl adenylate intermediate into an amino acid and an inorganic phosphoric acid or activity to hydrolyze aminoacyl tRNA into an amino acid and tRNA. The editing reaction active site exists in a domain independent of a domain containing the aminoacylation active site (Fukai, S. et. al., Cell, 2000, 103, 793-803.; Fukunaga, R., and Yokoyama, S., Nat Struct Mol Biol, 2005, 12, 915-922.; Nureki, O. et. al., Science, 1998, 280, 578-582.; Kotik-Kogan, O., Moor, N., Tworowski, D., and Safro, M., Structural basis for discrimination of L-phenylalanine from L-tyrosine by phenylalanyl-tRNA synthetase. Structure, 2005, 13, 1799-1807.; and Ribas de Pouplana, L., and Schimmel, P., Two classes of tRNA synthetases suggested by sterically compatible dockings on tRNA acceptor stem. Cell, 2001, 104, 191-193.). The domain is called an editing reaction domain. Specifically, each of these aaRSs strictly recognizes only a single amino acid by recognizing the separate properties (size, hydrophilicity, hydrophobicity) of an amino-acid side chain by the two reaction sites (Fukai, S. et. al., Cell, 2000, 103, 793-803.).
It should be noted that each of the aaRSs other than the aforementioned seven types of aaRS each of which has an editing reaction active site does not have an editing reaction site, and as such, recognizes a single amino acid only by an aminoacylation active site (Fersht, A. R., Shindler, J. S., and Tsui, W. C., Probing the limits of protein-amino acid side chain recognition with the aminoacyl-tRNA synthetases. Discrimination against phenylalanine by tyrosyl-tRNA synthetases. Biochemistry, 1980 19, 5520-5524.).
Incidentally, an aaRS mutant is produced by substituting an amino-acid residue of a substrate recognition site of a wild-type aaRS. For example, TyrRS is the first aaRS that succeeded in alteration for introduction of an unnatural amino acid. Further, TyrRS has a large amino-acid-binding pocket for recognizing a comparatively large amino acid, tyrosine, and presently has the largest number of mutants specific to unnatural amino acids (International Publication No. 2003/014354 Pamphlet; and Wang, L., Xie, J., and Schultz, P. G., Expanding the genetic code. Annu Rev Biophys Biomol Struct, 2006, 35, 225-249.). For example, there has been a report on a tyrosyl-tRNA synthetase (hereinafter referred to as “TyrRS”) mutant. Specifically, in International Publication No. 2003/014354 Pamphlet, known examples of a mutant that recognizes 3-iodotyrosine, which is an unnatural amino acid, include an Escherichia coli-derived TyrRS mutant and a Methanocaldococcus jannaschii-derived TyrRS mutant (MjIYRS).